CN108641000A

CN108641000A - The double target spot CAR-T therapy vectors and its construction method of liver cancer and application

Info

Publication number: CN108641000A
Application number: CN201810384082.2A
Authority: CN
Inventors: 田晓丽
Original assignee: Shanghai Yi Hao Biotechnology Co Ltd
Current assignee: Shanghai Yi Hao Biotechnology Co Ltd
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2018-10-12

Abstract

The present invention relates to the double target spot CAR T therapy vectors for providing liver cancer and its construction method and applications.Double target spot CAR T therapy vectors are made of Lentiviral pCDH EF1 α MCS (PGK Puro) and CAR structure two parts.The CAR structures are specially Igkappa iRGD CD133scFv-(G₄S)₅‑GPC3V_H‑(G₄S)₃‑GPC3V_LscFv‑CD8α‑CD28‑CD137‑CD3ζ‑T2A‑CCL19.The T cell containing the therapy vector, i.e. CAR T cells (Chimeric Antigen Receptor T Cell) are obtained with viral mode of infection, it, can targets identification and killing liver cancer cells by expressing CAR structures.

Description

The double target spot CAR-T therapy vectors and its construction method of liver cancer and application

Technical field

The present invention relates to a kind of CAR-T therapy vectors, more particularly, to the double target spot CAR-T therapy vectors and its structure of liver cancer Construction method and application.

Background technology

Liver cancer is common one of malignant tumour, it is clinical at present mainly take based on operation, intervention, local radiotherapy and chemotherapy Supplemented by comprehensive treatment, but there are high recurrence rate, the short problem of life span, and when most patients are medical be developed into It postpones or exists and be far apart organ metastasis and above-mentioned treatment can not be carried out.In recent years, immune biotherapy is considered as most being hopeful Non-operative treatment strategy.

LAK cells, DC, CIK cell are all once as the immunotherapeutic of tumour, but any of the above therapy lacks Weary specific recognition tumor associated antigen (tumor associated antigen, TAA) and killing specific tumors target cell Ability；And the process of cancer immunoediting can make the major histocompatibility complex (main of target cell surface Histocompatibility complex, MHC) decline in tumor cell surface expression, immunologic escape is formed, tumour cell is made T cell attack can successfully be hidden.

CAR-T cell therapies are artificially to be overexpressed to identify specific tumour on T cell surface by technique for gene engineering The single-chain variable fragments of surface antigen, to keep the target that T cell identification specific antigen, killing express the antigen thin Born of the same parents.Simultaneously as CAR-T cells are to identify antigen pattern using antibody, therefore not by major histocompatibility complex (main Histocompatibility complex, MHC) limitation.In recent years, CAR-T cells achieve in neoplastic hematologic disorder treatment Exciting effect, such as can to the complete incidence graph (CR) of late recurrent refractory childhood acute lymphoblastic leukemia (ALL) treatment Reach 90%, 50% or more is reached to the CR of chronic lymphocytic leukemia (CLL) and partial B cell lymthoma.It is real in treatment In terms of body tumor, safety and validity have been confirmed.

Chinese patent CN107557388A disclose the slow virus carrier prepared for CAR-T cells and its construction method and Application in preparing antitumor cell product, while the method for having also set up fluorescence quantitative PCR detection virus titer, for clinic Using providing examination criteria.

Chinese patent CN107446938A discloses a kind of selectively targeted receptor protein MAGEA and its CAR-T carriers Construction method, the construction method include first extracting the RNA of the monoclonal hybridoma of anti-osteosarcoma, then using monoclonal The specific primer of antibody carries out RT-PCR, and the variable region DNA fragmentation of heavy chain of antibody and light chain is cloned into carrier T, is sequenced, The receptor protein MAGEA of specific recognition tumour cell is obtained, and its expressed sequence is cloned on CAR-T carriers, is obtained special The novel C AR-T carriers of opposite sex targeting MAGEA.The invention can be the entity tumor of CAR-T cell therapies, especially in bone and flesh Effective target is provided in the immunization therapy of tumor.

Chinese patent CN107325185A discloses a kind of bis- targeting chimeric antigens of the PSCA based on OCTS-CAR and PDL1 Receptor, encoding gene, OCTS-CAR-T recombinant expression carriers and its construction method and application.The bis- targetings of the anti-PSCA and PDL1 are embedding It includes the CD8 leader membrane receptors signal peptide being sequentially connected in series, double antigen binding domains, CD8 Hinge chimeric to close antigen receptor Receptor hinge, CD8 Transmembrane Chimerical receptors transmembrane region, CD28 Chimerical receptors costimulating factor, OX40 Chimerical receptors Costimulating factor and TCR Chimerical receptor t cell activations domain, wherein double antigen binding domains include with series connection or corner connection side The heavy chain VH and light chain VL of PSCA and PDL1 single-chain antibodies of formula connection, antibody inner hinge Inner-Linker and single-stranded anti- Hinge Inter-Linker between body.In addition, the gene for encoding the bis- targeting Chimeric antigen receptors of the anti-PSCA and PDL1 is additionally provided, Recombinant expression carrier and its construction method and application.

Above-mentioned patent is the correlative study about CAR-T cell therapies, but lacks carry out double targets for liver cancer at present The carrier of point CAR-T treatments or correlative study.

Invention content

It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide double target spot CAR-T of liver cancer Therapy vector and its construction method and application.

The purpose of the present invention can be achieved through the following technical solutions：

First purpose of the invention：

A kind of Chimeric antigen receptor, i.e. CAR are provided, CAR structures include CD133 single-chain antibodies, GPC3 single-chain antibodies and become Change the factor tri- specificity structures of CCL19.

Wherein, CD133 single-chain antibodies structure is the specificity for the CD133 designs of liver cancer cells surface tumours related antigen Antigen binding domain, CD133 single-chain antibodies nucleotide sequence is as shown in SEQ ID NO.2.

Wherein, GPC3 single-chain antibodies structure is that the specificity designed for liver cancer cells surface tumours related antigen GPC3 resists Former binding domain, by GPC3 single-chain antibody heavy chains V_H、Inter-Linker(G₄S)₃, GPC3 single-chain antibody light chains V_LThree parts are constituted； Wherein, GPC3 single-chain antibodies heavy chain V_HNucleotide sequence as shown in SEQ ID NO.4.The nucleotide sequence of Inter-Linker As shown in SEQ ID NO.5.GPC3 single-chain antibody light chains V_LNucleotide sequence as shown in SEQ ID NO.6.

Chemokines CC CL19 chemotactic T cells infiltrate tumor tissues, and mediate tumor is immune, while inhibiting that suppression is immunized in tumour The release of the factor processed promotes Immune Cell Antigens to offer to act on, and inhibits vascularization indirectly, the final growth for inhibiting tumour； Chemokines CC CL19 nucleotide sequences are as shown in SEQ ID NO.12.

In addition, Recent study is found, CCR7 receptors in Expression In Hepatocellular Carcinoma, CCL19 can directly with CCR7 receptor knots It closes, access inhibits tumor proliferation, migration and invasion after activated receptor.

Further, CAR structure compositions are Igkappa-iRGD-CD133scFv- (G₄S)₅-GPC3V_H-(G₄S)₃- GPC3V_LscFv-CD8α-CD28-CD137-CD3ζ-T2A-CCL19。

Wherein iRGD structures can express iRGD peptides, and nucleotide sequence is as shown in SEQ ID NO.1, Kazuki Sugahara and its colleague once reported that the peptide of iRGD was a kind of peptide penetrating tumour, when iRGD is as a kind of individual object When matter is injected together with antitumor drug, drug delivery and antitumor activity can be greatly enhanced.

CD133 single-chain antibodies are expressed as CD133scFv in structure.

(G₄S)₅The hinge Inner-Linker between single-chain antibody, nucleotide sequence is as shown in SEQ ID NO.3.

GPC3 single-chain antibody heavy chains V_HIt is expressed as GPC3V in the structure_H, Inter-Linker is expressed as in the structure (G₄S)₃, GPC3 single-chain antibody light chains V_LIt is expressed as GPC3V in the structure_LscFv。

CD8 α are transmembrane region, connect extracellular antigen binding domain and intracellular signal domain, can be by CAR Structure anchors in T cell film On, nucleotide sequence is as shown in SEQ ID NO.7.

CD28-CD137 is costimulation structural domain, and the generation of transduction proliferation signal and inducing cytokine inhibits tumour life Long, CD28 nucleotide sequences are as shown in SEQ ID NO.8, and CD137 nucleotide sequences are as shown in SEQ ID NO.9.

CD3 ζ are signal transduction domain, when extracellular region and target antigen combination, will conduct TCR sample signals to intracellular, swash T cell living, plays the effect of targeting killing tumor cell, nucleotide sequence is as shown in SEQ ID NO.10.

T2A is that linker- is found in bright tetra- precursor virus of arteries and veins thosea siensis β (Thosea asigna), and effect is to make a list One segment coding protein, its advantage is that two genes (ORF) can be connected to become an ORF by T2A polypeptides, mRNA is translated into One fusion protein, the fusion protein can be identified the proteolytic cleavage of T2A into two albumen, nucleotide sequence such as SEQ ID Shown in NO.11.

Second purpose of the invention：

A kind of double target spot CAR-T therapy vectors of liver cancer are provided, the Chimeric antigen receptor of first goal of the invention is included, with And PUC19 plasmids.The Chimeric antigen receptor of first goal of the invention is present on PUC19 plasmid vectors.

Third purpose of the present invention：

A kind of double target spot CAR-T therapy vectors of liver cancer, including Lentiviral pCDH-EF1 α-MCS- are provided (PGK-Puro) and CAR.

Wherein, Lentiviral pCDH-EF1 α-MCS- (PGK-Puro), structure is as shown in Figure 1.Pass through Snap Gene softwares analyze the carrier and search pertinent literature it is found that pCDH-EF1 α-MCS- (PGK-Puro) are bis- with EcoRI and NotI Digestion Insert Fragment.The expression vector includes：EF1 α promoters-are mammalian cell specificity promoters, driving capability compared with By force；Multiple cloning sites (MCS)-include multiple restriction enzyme sites (restriction site), are that foreign gene is inserted into Position；The polyA tailing efficiency of mRNA can be improved in WPRE elements-, improves the expression efficiency of metastatic gene；SV40polyA sequences- Transcription can effectively be terminated and add PolyA tails for the mRNA of transcription；Hybrid RSV/5 ' LTR- contain promoter and enhancer Equal controlling elements, make its high-caliber expression overall length virus transcription object in 293T cells；Genetic element (cPPT, gag, env, LTRs)-for packing, transduceing and steadily will be in the genomic DNA of expressing viral structural integrity to host；SV40origin- Plasmid is set to stablize proliferation in incasing cells.

The Lentiviral, which can be used as, makes target gene in nearly all mammalian cell include non-dividing cell With the most effective carrier expressed in dividing cell, can hold carry exogenous genetic fragment it is big, transfection efficiency is higher, also can to T cell Reach satisfied transfection.

4th purpose of the invention：A kind of construction method of double target spot CAR-T therapy vectors of liver cancer is provided.

It by above-mentioned CAR structures according to gene order, is synthesized using standard biologic synthetic method, the CAR after synthesis is deposited It is on PUC19 plasmid vectors；Lentiviral pCDH-EF1 α-MCS- (PGK-Puro) are purchased from SBI.By PUC19 plasmids Carrier and Lentiviral are all made of ECoR, I Not double digestions, and digestion products are detached by agarose gel electrophoresis, are returned Receive purpose band, obtain the concentration of carrier and target fragment, by the two according to molar ratio be 1:5 are attached conversion, and plasmid carries It takes, it is final to obtain the recombinant plasmid containing specific CAR structures.

5th purpose of the invention：The application for providing double target spot CAR-T therapy vectors of liver cancer, that is, provide a kind of CAR-T Cell.

The therapy vector is subjected to slow virus packaging using three plasmid packaging systems, group becomes PSPAX2, pMD2G, liver cancer Double target spot CAR-T therapy vectors, using 293T cells as the incasing cells of slow virus, after collecting culture 48h, 72h respectively Virus liquid, this virus liquid is concentrated, and survey virus titer, finally with MOI=20 infect T cell, you can obtain CAR-T it is thin Born of the same parents.

Content of this three kinds of plasmids in system (6cm wares system) is respectively 4 μ g, 0.5 μ g, 3 μ g.

In the present invention, CAR structures include CD133 single-chain antibodies, GPC3 single-chain antibodies and Chemokines CC CL19 tri- special Property structure；CD133 single-chain antibodies, GPC3 single-chain antibodies be according to liver cancer cells surface can express tumor associated antigen CD133, The specificity structure that GPC3 is determined, two strain specific antibodies of Anti-CD133, Anti-GPC3 of the structure representation are responsible for identification and swell Tumor related antigen (tumor associated antigen, TAA), on the one hand can targeting killing liver cancer cells；On the other hand, It assigns T cell new antigentic specificity, can effectively avoid tumour cell MHC expression and lower this Immune escaping mechanism.Chemotactic because Sub- CCL19 can induce in immunocyte infiltration to tumor tissues and play immune function.When T cell is obtained by viral mode of infection The carrier and after expressing the CAR structures, T cell can targeting identify and kill liver cancer cells.It, can by expressing CAR structures Targets identification and killing liver cancer cells.

Compared with prior art, the present invention has the following advantages and beneficial effects：

Since the T cell can express the antibody of tumor associated antigen GPC3, CD133 for liver cancer cells surface simultaneously Anti-GPC3, Anti-CD133 so that its identification range greatly increases, wider for the hazard boundary of liver cancer cells；Meanwhile It can be fitted into the CAR-T cells of single antigen receptor to avoid multiple infusion, not only alleviate the injury to patient, can also mitigate Its economic pressures；In addition, CAR structures mentioned in the present invention impart the stronger proliferative of T cell, lasting vitality, and Tumor tissues are infiltrated by Chemokines CC CL19 chemotactic T cells, inhibits the release of immune factor in tumor, so that T cell is overcome swollen Tumor local immunosuppression microenvironment and break host immune tolerance status, quickly establish tumour immunity and eliminates small-sized or large-scale swollen Tumor is born.

Description of the drawings

Fig. 1 is Lentiviral pCDH-EF1 α-MCS- (PGK-Puro) structural schematic diagram；

Fig. 2 is cytotoxicity analysis detection technique flow chart；

Fig. 3 is the Western blot testing results of targeting antigen CD133 expressions in SK-HEP-1 cell lines；

Fig. 4 is agarose gel electrophoresis knot of pCDH-EF1 α-MCS- (PGK-Puro) plasmids after EcoR, I Not digestions Fruit.

Specific implementation mode

Material source in following embodiment is described as follows：

1. slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro), slow virus packaging plasmid pMD2G, vector plasmid PSPAX2 is purchased from SBI；Slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro) structure is as shown in Figure 1.

2.CAR structure sequences are designed by the Shanghai bio tech ltd Yi Hao, by raw work bioengineering (Shanghai) share Co., Ltd synthesizes, and is preserved with PUC19 plasmid forms；

3. restriction endonuclease EcoR I, Not I are purchased from NEB；

4.T4DNA ligase、Free H₂O is purchased from precious biology；

5. competent cell is purchased from Trans；

6. plastic recovery kit, the small extraction reagent kit of plasmid is purchased from Tiangeng biochemical technology Co., Ltd；

7.293T cells, SK-HEP-1 cells are purchased from Chinese Academy of Sciences's cell bank；

8.FBS, DMEM, 1640 culture mediums, PBS, Opti-MEM, lipofectamine 2000 are purchased from Gibco；

9. cell pyrolysis liquid RIPA, protease inhibitors is purchased from the green skies；

10.BCA protein quantifications kit, the super quick colour reagent box of ECL chemiluminescences, Tween-20,5 × SDS-PAGE eggs White sample-loading buffer, HRP-labeled Goat Anti-Rabbit IgGHRP-labeled Goat Anti-Mouse IgG Purchased from assist sage；

11. electrophoresis liquid, fast-turn construction liquid, glue are purchased from Tanon；

12. skimmed milk power is purchased from BD；

13.PVDF films are purchased from Millipore；

14. membrane regeneration liquor is purchased from the bio tech ltd Suo Laibao；

15.Anti-CD3zeta antiboby are purchased from abcam；

16.Beta Actin Antibody are purchased from Wuhan Sanying Bio-Technology Co., Ltd.；

17. albumen Marker is purchased from Thermo；

18. high-pressure steam sterilizing pan is pacified purchased from Shanghai Shen；

19. air dry oven is purchased from Shanghai Ba Jiu Industrial Co., Ltd.s；

20.ProFlex PCR instruments are purchased from ABI；

21.Multiskan GO microplate reader+uDrop ultra micros template, flow cytometer are purchased from ThermoFisher；

22.ST16R tabletop refrigerated centrifuges, Fresco microcentrifuges are purchased from ThermoFisher；

23.HE120 Horizontal electrophoresis tank, Tanon gel imagers are purchased from Tanon；

24. Biohazard Safety Equipment, CO₂4 DEG C of directly-heated type cell incubator, double door refrigerators are purchased from ThermoFisher；

25. pipettor, electronic suction assisting device are purchased from Eppendorf；

26. water isolation type constant incubator, constant-temperature shaking incubator are purchased from the permanent Science and Technology Ltd. in Shanghai one；

The small-sized electrophoresis systems of 27.Bio-Rad Mini-PROTEAN Tetra Cell are purchased from Bio-Rad；

28. Olympus microscope is purchased from Olympus；

29. oese, spreading rod Jie Te biofiltrations limited liability company；

30. syringe, 0.45 μm of filter membrane, each specification culture dish, culture bottle, porous culture plate, various specifications centrifuge tube Purchased from Corning；

A kind of Chimeric antigen receptor of the present invention, i.e. CAR, CAR structures include CD133 single-chain antibodies, GPC3 single-chain antibodies and Tri- specificity structures of Chemokines CC CL19.

CAR structure compositions are Igkappa-iRGD-CD133scFv- (G₄S)₅-GPC3V_H-(G₄S)₃-GPC3V_LscFv-CD8 α-CD28-CD137-CD3ζ-T2A-CCL19。

Wherein iRGD structures can express iRGD peptides, and nucleotide sequence is as shown in SEQ ID NO.1, Kazuki Sugahara and its colleague once reported that the peptide of iRGD was a kind of peptide penetrating tumour, when iRGD is as a kind of individual object When matter is injected together with antitumor drug, drug delivery and antitumor activity can be greatly enhanced.CD133 single-chain antibody structures are needles To the specific antigen binding domain of liver cancer cells surface tumours related antigen CD133 designs, CD133 single-chain antibody nucleotide sequences As shown in SEQ ID NO.2.GPC3 single-chain antibody structures are the spies for the GPC3 designs of liver cancer cells surface tumours related antigen Specific Antigen binding domain, by GPC3 single-chain antibody heavy chains V_H、Inter-Linker(G₄S)₃, GPC3 single-chain antibody light chains V_LThree Divide and constitutes；Wherein, GPC3 single-chain antibodies heavy chain V_HNucleotide sequence as shown in SEQ ID NO.4.The core of Inter-Linker Nucleotide sequence is as shown in SEQ ID NO.5.GPC3 single-chain antibody light chains V_LNucleotide sequence as shown in SEQ ID NO.6. CD133 single-chain antibodies are expressed as CD133scFv in structure.(G₄S)₅The hinge Inner-Linker between single-chain antibody, nucleosides Acid sequence is as shown in SEQ ID NO.3.GPC3 single-chain antibody heavy chains V_HIt is expressed as GPC3V in the structure_H, Inter-Linker exists (G is expressed as in structure₄S)₃, GPC3 single-chain antibody light chains V_LIt is expressed as GPC3V in the structure_LscFv.CD8 α are transmembrane region, even Extracellular antigen binding domain and intracellular signal domain are connect, can be by CAR Structure anchors on T cell film, nucleotide sequence such as SEQ ID Shown in NO.7.CD28-CD137 is costimulation structural domain, and the generation of transduction proliferation signal and inducing cytokine inhibits tumour life Long, CD28 nucleotide sequences are as shown in SEQ ID NO.8, and CD137 nucleotide sequences are as shown in SEQ ID NO.9.CD3 ζ are letter Number transduction domain will conduct TCR sample signals to intracellular, activate T cell, play targeting when extracellular region and target antigen combine The effect of property killing tumor cell, nucleotide sequence is as shown in SEQ ID NO.10.T2A is that linker- is found in the flat thorn of bright arteries and veins Tetra- precursor virus of moth β (Thosea asigna), effect is to make a single fragment coding protein, its advantage is that two genes (ORF) ORF can be connected to become by T2A polypeptides, mRNA translates into a fusion protein, which can be identified The proteolytic cleavage of T2A is at two albumen, and nucleotide sequence is as shown in SEQ ID NO.11.Chemokines CC CL19 chemotactic T cells Tumor tissues are infiltrated, mediate tumor is immune, while inhibiting the release of immunosuppressive factor in tumour, promotes immunocyte anti-indirectly Original offers to act on, and inhibits vascularization, the final growth for inhibiting tumour；Chemokines CC CL19 nucleotide sequences such as SEQ ID Shown in NO.12.In addition, Recent study is found, CCR7 receptors in Expression In Hepatocellular Carcinoma, CCL19 can directly with CCR7 receptors In conjunction with access inhibits tumor proliferation, migration and invasion after activated receptor.

Double target spot CAR-T therapy vectors of liver cancer, including Lentiviral pCDH-EF1 α-MCS- (PGK-Puro) And CAR.

Wherein, Lentiviral pCDH-EF1 α-MCS- (PGK-Puro), structure is as shown in Figure 1.Pass through Snap Gene softwares analyze the carrier and search pertinent literature it is found that pCDH-EF1 α-MCS- (PGK-Puro) are bis- with EcoRI and NotI Digestion Insert Fragment.The expression vector includes：EF1 α promoters-are mammalian cell specificity promoters, driving capability compared with By force；Multiple cloning sites (MCS)-include multiple restriction enzyme sites (restriction site), are that foreign gene is inserted into Position；The polyA tailing efficiency of mRNA can be improved in WPRE elements-, improves the expression efficiency of metastatic gene；SV40 polyA sequences It arrange-can effectively terminate transcription and add PolyA tails for the mRNA of transcription；Hybrid RSV/5 ' LTR- contain promoter and enhancing The controlling elements such as son make its high-caliber expression overall length virus transcription object in 293T cells；Genetic element (cPPT, gag, Env, LTRs)-for packing, transduceing and steadily will be in the genomic DNA of expressing viral structural integrity to host；SV40 Origin- makes plasmid stablize proliferation in incasing cells.The Lentiviral, which can be used as, makes target gene nearly all Mammalian cell includes the most effective carrier expressed in non-dividing cell and dividing cell, can hold load exogenous genetic fragment Greatly, transfection efficiency is higher, can also reach satisfied transfection to T cell.

The construction method of double target spot CAR-T therapy vectors of liver cancer：By above-mentioned CAR structures according to gene order, using normal Rule biological synthesis method is synthesized, and the CAR after synthesis is present on PUC19 plasmid vectors；Lentiviral pCDH- EF1 α-MCS- (PGK-Puro) are purchased from SBI.PUC19 plasmid vectors and Lentiviral are all made of ECoR I, Not I couple Digestion products are detached by agarose gel electrophoresis, recycle purpose band, obtain the concentration of carrier and target fragment by digestion, By the two according to molar ratio be 1:5 are attached conversion, and plasmid extraction is final to obtain the recombinant plasmid containing specific CAR structures.

The therapy vector is subjected to slow virus packaging using three plasmid packaging systems, group becomes PSPAX2, pMD2G, liver cancer Double target spot CAR-T therapy vectors, content of this three kinds of plasmids in system (6cm wares system) is respectively 4 μ g, 0.5 μ g, 3 μ g. Using 293T cells as the incasing cells of slow virus, the virus liquid after culture 48h, 72h is collected respectively, this virus liquid is dense Contracting, and virus titer is surveyed, T cell is finally infected with MOI=20, you can obtain CAR-T cells.

The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.

Embodiment

(1) plasmid extraction

The preparation method of LB liquid medium：Electronic balance weighs 5g fluid nutrient mediums dry powder in 500mL conical flasks, adds 100mL ultra-pure waters sterilize after masking foil sealing in high-pressure steam sterilizing pan, when being cooled to 40 DEG C~50 DEG C, with 1000:1 is added 0.2% ampicillin, after careful mixing, is transferred to spare in clean 500mL reagent bottles, condition of storage is 4℃。

The preparation method of LB solid mediums：Electronic balance weighs 5g solid mediums dry powder in 500mL conical flasks, adds 100mL ultra-pure waters sterilize after masking foil sealing in high-pressure steam sterilizing pan, when being cooled to 40 DEG C~50 DEG C, with 1000:1 is added 0.2% ampicillin, and after careful mixing, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after solidification, seals membrana oralis and is stored in 4 DEG C.

Glycerol stock is taken out respectively from -80 DEG C of refrigerators, and glycerol stock contains slow virus expression plasmid pCDH-EF1 α-MCS- (PGK- Puro) and PUC19 plasmids, take 5 μ L to be inoculated in 5mL LB liquid mediums (AMP resistances) respectively, respectively take 8 pipes, carry out respectively Label, in constant-temperature table 12~16h of shaken cultivation, condition is 37 DEG C, 250rmp.

The small extraction reagent kit of plasmid is purchased from Tiangeng biochemical technology Co., Ltd, and plasmid extraction is carried out by its specification.Specific behaviour Make as follows：Following cp3, P1, p2, p3, pw belong to the reagent inside mentioned reagent box, article No.：DP103-03

1) column equilibration：Adsorption column CP3,500 μ L equilibrium liquids BL, 12,000rpm, centrifugation 1min are outwelled useless in collecting pipe Liquid places back in adsorption column in collecting pipe.(processed pillar on the day of use)；

2) centrifuge tube is added in the bacterium solution for taking 1mL to be incubated overnight, and 12,000rpm, centrifugation 1min, as possible absorption supernatant are (repeatedly Bacterial sediment is collected into a centrifuge tube by centrifugation)；

3) 250 μ L solution P1, suspension mixing precipitation, if there is not thorough mixing are added into the centrifuge tube of bacterial sediment Fungus block can influence to crack, cause extracted amount and purity relatively low；

4) 250 μ L solution P2 are added, mild spins upside down 6-8 times, and thalline is made fully to crack.(it is gently blended, it should not Acutely concussion causes to be mixed with genomic DNA fragment in the plasmid of extraction in order to avoid interrupting genomic DNA.Bacterium solution should become clear at this time Bright sticky, 5min is not to be exceeded in the time used, in case plasmid is destroyed., may be excessive due to thalline if not becoming limpid, Cracking is not thorough, and should reduce biomass)；

5) 350 μ L solution P3 are added into centrifuge tube, leniently spins upside down 6-8 times, mixes well immediately, will go out at this time Existing white flock precipitate.12,000rpm, 10min is centrifuged, forms precipitation in centrifugation bottom of the tube at this time.(P3 should be mixed immediately after being added It closes, avoids generating localized precipitation.If there is minute white precipitation in supernatant, supernatant is taken after can centrifuging again)；

6) supernatant that previous step is collected is transferred in adsorption column CP3 (adsorption column is put into collecting pipe) with pipettor, Pay attention to trying not that precipitation is sucked out.12,000rpm, 60s is centrifuged, the waste liquid in collecting pipe is outwelled, adsorption column CP3 is put into collection Guan Zhong.

7) 600 μ L rinsing liquids PW are added into adsorption column CP3,12,000rpm, centrifugation 1min are outwelled useless in collecting pipe Adsorption column CP3 is put into collecting pipe by liquid.It is repeated once；

8) adsorption column CP3 is put into collecting pipe, 12,000rpm, centrifuges 2min, rinsing liquid remaining in adsorption column is gone It removes, is placed in and is placed at room temperature for 2min, thoroughly to dry remnants；

9) adsorption column CP3 is placed in a clean centrifuge tube, 60 μ L Free H is added dropwise to adsorbed film centre₂O, room Temperature places 2min, 12000rpm, centrifuges 2min, plasmid is collected into centrifuge tube, slow virus expression plasmid pCDH- is respectively obtained EF1 α-MCS- (PGK-Puro) and PUC19 plasmids.Microplate reader surveys production concentration：Slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro) a concentration of 358ng/ μ L；PUC19

Plasmid be 100.3ng/ μ L, finish writing label, be stored in -20 DEG C it is spare.

(2) digestion, connection, conversion

1. digestion

Slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro), PUC19 plasmids are carried out with following system simultaneously EcoRI, NotI double digestion, 37 DEG C of water-bath 1-2h；

pCDH-EF1α-MCS-(PGK-Puro)/PUC19

Sample-adding amount is determined according to plasmid concentration, and plasmid is 2 μ g in system

2. agarose gel electrophoresis

Slow virus expression plasmid pCDH-EF1 α-MCS- (PGK-Puro) digestion products need 0.8% Ago-Gel： Agarose 0.16g is weighed in 100mL conical flasks, is added in 1 × TAE of 20mL, micro-wave oven dissolves by heating, and is cooled to 50 DEG C At~60 DEG C, the nucleic acid dye (glod view) of 2 μ L is added, shakes up, is poured into and has been plugged in comb gel slab, solidification is spare.

PUC19 plasmid enzyme restriction products need 1.0% Ago-Gel：Agarose 0.2g is weighed in 100mL conical flasks, It being added in 1 × TAE of 20mL, micro-wave oven dissolves by heating, and when being cooled to 50 DEG C~60 DEG C, the nucleic acid dye of 2 μ L is added, shakes up, It is poured into and has been plugged in comb gel slab, solidification is spare.

After glue cooled and solidified, 6 × Loading buffer are added according to final volume in plasmid enzyme restriction product, piping and druming is mixed It is even, it sequentially adds in loading hole.It is put into electrophoresis tank, 90V, 30min observe and record result with gel imager.Agarose is solidifying Gel electrophoresis result pCDH-EF1 α-MCS- (PGK-Puro) are as shown in Figure 4.

3. glue recycles

Glue recycling is carried out with Tiangeng Ago-Gel QIAquick Gel Extraction Kit specification, concrete operation step is as follows：It adsorbs below Column CA2, solution PN, it is reagent in mentioned reagent box, article No.：DP209-02

1) column equilibration step：Into adsorption column CA2, (adsorption column is put into collecting pipe) is added 500 μ L equilibrium liquids BL, and 12, 000rpm (~13,400g) centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.It (please use The same day processed pillar)；

2) single target DNA band is cut from Ago-Gel and (cuts off redundance as possible) be put into it is clean from In heart pipe, weight is weighed；

3) equimultiple bulk solution PN is added into blob of viscose, and (if gel weight is 0.1g, volume can be considered 100 μ l, then adds Enter 100 μ l PN solution), 56 DEG C of water-baths are placed, and constantly centrifuge tube are leniently spun upside down therebetween, to ensure that blob of viscose fully dissolves. If there is not molten blob of viscose, it can continue to place a few minutes or add some sol solutions again, until blob of viscose is completely dissolved (if blob of viscose Volume it is excessive, can blob of viscose be cut into fragment in advance).Pay attention to：For recycling<The small fragment of 300bp can be completely molten in addition PN The isopropanol of 1/2 blob of viscose volume is added after glue to improve the rate of recovery；Solution temperature is preferably down to room by blob of viscose after being completely dissolved Warm upper prop again, because ability of the adsorption column in room temperature in conjunction with DNA is stronger；

4) previous step acquired solution is added in an adsorption column CA2 (adsorption column is put into collecting pipe), is placed at room temperature for 2min, 12,000rpm (~13,400g) centrifuge 30-60s, outwell the waste liquid in collecting pipe, adsorption column CA2 is put into collecting pipe In.Pay attention to：Absorption column volume is 800 μ L, if sample volume is more than 800 μ L and can be added portionwise；

5) 600 μ L rinsing liquids PW (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column CA2, 12,000rpm (~13,400g) centrifuge 30-60s, outwell the waste liquid in collecting pipe, adsorption column CA2 is put into collecting pipe.Note Meaning：If the DNA of recycling is the experiment for salt density value, such as blunt end cloning experiment or direct Sequencing, it is proposed that after PW is added 2-5min is stood to centrifuge again；

6) repetitive operation step (5)；

7) adsorption column CA2 is put back in collecting pipe, 12,000rpm (~13,400g) centrifuge 2min, eliminate rinsing as possible Liquid.Adsorption column CA2 is placed in and is placed at room temperature for several minutes, is thoroughly dried, to prevent remaining rinsing liquid from influencing the reality of next step It tests.

Pay attention to：The residual of ethyl alcohol can influence subsequent enzyme reaction (digestion, PCR etc.) experiment in rinsing liquid；

8) adsorption column CA2 is put into a clean centrifuge tube, 30 μ L Free is vacantly added dropwise to adsorbed film centre position H2O is placed at room temperature for 2min.12,000rpm (~13,400g) centrifuge 2min and collect DNA solution, microplate reader detectable concentration：pCDH- It is a concentration of 4ng/ μ L of 15.9ng/ μ L, CAR structures that concentration is recycled after EF1 α-MCS- (PGK-Puro) digestion.

4. connection, conversion

1) recycling segment is pressed into CAR structures:PCDH-EF1 α-MCS- (PGK-Puro)=5:1 molar ratio is attached, 16 DEG C, 4h is connected in ProFlex PCR instruments.

Linked system is as follows：

Subsequent operation carries out in superclean bench.

Wherein, CAR structure compositions are Igkappa-iRGD-CD133scFv- (G₄S)₅-GPC3V_H-(G₄S)₃- GPC3V_LscFv-CD8α-CD28-CD137-CD3ζ-T2A-CCL19.IRGD structures can express iRGD peptides, and nucleotide sequence is such as Shown in SEQ ID NO.1, Kazuki Sugahara and its colleague once reported that the peptide of iRGD was a kind of to penetrate tumour Peptide can greatly enhance drug delivery and antitumor when iRGD is injected as a kind of individual substance together with antitumor drug Activity.CD133 single-chain antibody structures are the specific antigen combinations for the CD133 designs of liver cancer cells surface tumours related antigen Domain, CD133 single-chain antibodies nucleotide sequence is as shown in SEQ ID NO.2.GPC3 single-chain antibody structures are to be directed to liver cancer cells table The specific antigen binding domain of face tumor associated antigen GPC3 designs, by GPC3 single-chain antibody heavy chains V_H、Inter-Linker (G₄S)₃, GPC3 single-chain antibody light chains V_LThree parts are constituted；Wherein, GPC3 single-chain antibodies heavy chain V_HNucleotide sequence such as SEQ Shown in ID NO.4.The nucleotide sequence of Inter-Linker is as shown in SEQ ID NO.5.GPC3 single-chain antibody light chains V_LCore Nucleotide sequence is as shown in SEQ ID NO.6.CD133 single-chain antibodies are expressed as CD133scFv in structure.(G₄S)₅For single-chain antibody Between hinge Inner-Linker, nucleotide sequence is as shown in SEQ ID NO.3.GPC3 single-chain antibody heavy chains V_HTable in the structure It is shown as GPC3V_H, Inter-Linker is expressed as (G in the structure₄S)₃, GPC3 single-chain antibody light chains V_LIt is expressed as in the structure GPC3V_LscFv.CD8 α are transmembrane region, connect extracellular antigen binding domain and intracellular signal domain, can be by CAR Structure anchors in T cell On film, nucleotide sequence is as shown in SEQ ID NO.7.CD28-CD137 is costimulation structural domain, and transduction proliferation signal simultaneously induces The generation of cell factor inhibits tumour growth, and CD28 nucleotide sequences are as shown in SEQ ID NO.8, and CD137 nucleotide sequences are such as Shown in SEQ ID NO.9.CD3 ζ are signal transduction domain, when extracellular region and target antigen combination, will conduct TCR samples to intracellular Signal activates T cell, plays the effect of targeting killing tumor cell, nucleotide sequence is as shown in SEQ ID NO.10.T2A It is found in bright tetra- precursor virus of arteries and veins thosea siensis β (Thosea asigna) for linker-, effect is to make a single fragment coding Protein, its advantage is that two genes (ORF) can be connected to become an ORF by T2A polypeptides, mRNA translates into a fusion egg In vain, which can be identified the proteolytic cleavage of T2A into two albumen, and nucleotide sequence is as shown in SEQ ID NO.11. Chemokines CC CL19 chemotactic T cells infiltrate tumor tissues, and mediate tumor is immune, while inhibiting immunosuppressive factor in tumour Release promotes Immune Cell Antigens to offer to act on, and inhibits vascularization indirectly, the final growth for inhibiting tumour；Chemotactic factor (CF) CCL19 nucleotide sequences are as shown in SEQ ID NO.12.CAR structure sequences are designed by the Shanghai bio tech ltd Yi Hao, It is synthesized by Sangon Biotech (Shanghai) Co., Ltd., is preserved with PUC19 plasmid forms.

2) it takes out competent cell from -80 DEG C of refrigerators to melt on ice, taking out solid culture from 4 DEG C is based on 37 DEG C of incubators Preheating；

3) connection product is transferred in 1.5mL centrifuge tubes；

4) it is slowly added to 50 μ L competent cells thereto, agitates by adding, makes the two mixing；

5) after the centrifuge tube in 3 being put in 30min on ice, the heat shock 90s in 37 DEG C of water-baths；

6) it after heat shock, takes out be placed in 2min on ice immediately；

7) 950 μ L fluid nutrient mediums are added thereto, in constant-temperature table 40~60min of culture, condition is 37 DEG C, 200rmp；

8) 6000rpm centrifuges 2min, discards most of supernatant, stay 40~60 μ L；

9) resuspended bacterium solution is blown and beaten with pipette tips, bacterium solution is dripped on the solid medium of preheating, is marked, 37 DEG C of incubator mistakes Night；

(3) upgrading grain, digestion verification, sequencing

1) for picking part bacterium colony in 5mL fluid nutrient mediums, constant-temperature table carries out Zengjing Granule, and condition is 37 DEG C, 250rmp cultivates 12~16h；

2) plasmid extraction, and measured concentration are carried out with the small extraction reagent kit of plasmid purchased from Tiangeng bio tech ltd； Each 500 μ L that take out are used as fungi preservation, are stored in -80 DEG C in 1.5mLEp pipes before plasmid extraction；

3) gained plasmid is subjected to EcoRI, NotI double digestion, 37 DEG C of water-bath 1-2h.Digestion system is as follows：

Structure containing CAR pCDH-EF1 α-MCS- (PGK-Puro) plasmid

Sample-adding amount is determined according to plasmid concentration, and plasmid is 1 μ g in system

4) agarose gel electrophoresis.The Ago-Gel 50ml of method configuration 0.8% as above, and carry out electrophoresis, 90V, 20min observes and records result with gel imager；

5) take 1 μ g that Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced the correct plasmid of band, band is abnormal Plasmid and the bacterium solution of retention abandon.The correct plasmid of sequencing result is stripped, the plasmid of sequencing result mistake and Its bacterium solution abandons.

(4) preparation of concentrating virus liquid

1. virus liquid is collected

Slow virus packaging is carried out using three plasmid packaging systems.Three plasmids are respectively the slow virus expression containing CAR structures Plasmid pCDH-EF1 α-MCS- (PGK-Puro), slow virus packaging plasmid pMD2G, vector plasmid PSPAX2.Cell is that 293T is thin Born of the same parents.

Specific implementation step is as follows：

1) interior for 24 hours before transfecting to carry out bed board：It is typically chosen cell of the passage number within 3 generations, it is close according to cell growth Degree and state adjust cell density, and growth conditions are good in 10cm wares, and waste liquid is abandoned in the 293T cells suction that stand density reaches 80%；

2) thereto slowly adherent addition 3mLPBS to wash cell；

3) it inhales after abandoning waste liquid, slow adherent addition 1mL pancreatin digests 2min in constant incubator；

4) the DMEM complete mediums (10%FBS) of 3mL are added into the cell digested to terminate digestion, and are transferred to 800rpm centrifuges 3min in 15mL centrifuge tubes；

5) it discards supernatant, into cell precipitation plus cell is resuspended in 5mL DMEM complete mediums (10%FBS), slowly blows and beats Mixing；

6) 6 6cm wares are taken, and add 5mL DMEM complete mediums (10%FBS)；

7) 720 μ L cell suspensions are added into each 6cm wares, careful mixing makes cell need completely adherent and adherent uniform；

8) for density to be grown up to 60~90%, cell state is good, you can carries out viral packaging；

9) according to transfectional cell quantity, by three plasmid mixed liquors needed for each 6cm wares of following proportional arrangement

3 μ g of recombinant plasmid

pMD2G 0.5μg

PSPAX2 4μg

The dosage for needing to be added is determined according to each plasmid concentration.The dosage that this need to be added is as follows：

Recombinant plasmid (124ng/ μ L) 24.2 μ L

pMD2G(436ng/μL) 1.15μL

PSPAX2(246ng/μL) 14.5μL

2mL Opti-MEM are added into plasmid mixed liquor, are stored at room temperature 5min；

10) lipofectamine 2000 (4 DEG C preservation) mixed liquor need to be separately configured, needed for each 6cm wares Lipofectamine 2000 is 2 μ L/ μ g, tri- plasmid mixed liquors, and 2mLOpti-MEM is slowly added dropwise thereto, is stored at room temperature 5min；

11) above (9) and (10) are mixed in a pipe, the static 20min of room temperature；

12) the 293T cells completed are taken out, waste liquid, and careful adherent addition 1mLDMEM culture mediums are abandoned in suction, are prevented thin Born of the same parents float；

13) will mixed liquor in (11) it is careful it is adherent be added in the cell in (12), prevent cells float, after mixing, after Continuous culture 72h；

14) culture supernatant after collection 72h, it is spare by 0.45 μm of membrane filtration；

2. virus liquid concentrates

1) 5 × PEG8000NaCl is prepared：Weigh NaCl 8.766g；It is pure that PEG8000 50g are dissolved in 200mL Milli-Q In water；121 DEG C, moist heat sterilization 30min；It is stored in 4 DEG C；

2) per the filtered initial liquid of virus of 30mL, 5 × PEG-8000NaCl mother liquors 7.5mL is added；

3) every 20~30min mixing is primary, carries out 3-5 times altogether；

4) it stands overnight for 4 DEG C；

5) 4 DEG C, 4000g, 20min is centrifuged；

6) it inhales and abandons supernatant, stand 1~2min of pipe, siphon away residual liquid；

7) per the filtered initial liquid of virus of 30mL, 500 μ LPBS dissolving slow virus precipitations are added；

8) viral suspension after collecting is distributed into 50 every part of μ L, is stored in production tube.Be stored in after broken dry ice quick-frozen- 80 DEG C spare (avoiding concentrating virus liquid multigelation).

3. virus titer measures

1) the good 293T cells of growth conditions are taken, are counted after digestion, by 2 × 10⁵Cells/well uniformly spreads cell to 24 Porocyte culture plates, 6~10h of culture are adherent to cell；

2) the concentrating virus liquid of various concentration is added in cells and supernatant into 24 orifice plates, gently pats 24 hole edges of boards Edge makes virus liquid be uniformly mixed with culture solution, and 48h is infected in incubator；

3) after infecting 48h, digest and collect cell, Flow cytometry positive cell percentage, and according to following public affairs Formula calculates virus titer, unit TU/mL：

The virus liquid volume (μ L) of T=(cell number × positive cell percentage × 1000 when bed board)/addition

(5) virus liquid infects T cell

1.PBMC is detached

1) about 6mL people periphery new blood is collected with the vacuum blood collection tube containing heparin；

2) it dilutes：Isometric PBS is added at room temperature, gently blows and beats mixing；

3) it is loaded：50mL centrifuge tubes are taken, draw 6mL Ficoll (lymphocyte separation medium) in centrifuge tube, (Ficoll Volume ratio with blood before dilution is 1:1), pipe tilt 45 °, by the blood after dilution at Ficoll ullages about 1cm edge Tube wall is added slowly to above Ficoll；

4) it centrifuges：18-20 DEG C, 2000rpm, 30min, points four layers from tube bottom to liquid level after centrifugation, be followed successively by red blood cell and Granulocyte layer, layering liquid layer, mononuclearcell layer, plasma layer；

5) it recycles：Pipette is inserted directly into mononuclearcell layer (or first sucking the blood plasma on upper layer), is gently sucked out single A nucleus layer, is put into new centrifuge tube；

6) it washs：Be added to be less than 3 times of PBMC (peripheral blood mononuclear cells) volume PBS, 18-20 DEG C, 2000rpm, 10min, twice；

7) cell count：Supernatant is abandoned, adds 1mLRPMI-1640 culture mediums (containing 10% fetal calf serum), blows and beats mixing, prepare At PBMC cell suspensions.It is counted using blood cell counting plate：Add after taking a drop PBMC suspensions and a 2% trypan blue dye liquor mixing of drop In blood cell counting plate, 4 big lattice inner cell sums are counted under the microscope.Cell number/mL=4 block plaid total number of cells/4 ×10⁴× 2 (extension rates)

The activation and slow-virus infection of 2.T cells

1) cell density is adjusted to 1 × 10⁶Cell factor and antibody complex (final concentration 100U/mL is added in cell/mL IL-2,100ng/m L Anti-CD3 (OKT3), 250ng/mL Anti-CD28, continuously cultivate 48h；

2) according to MOI=20, required virus quantity is calculated.Calculation formula is as follows：

Required virus quantity (mL)=(MOI × cell quantity)/virus titer

3) after -80 DEG C of refrigerators take out virus, melt in 37 DEG C of water-baths rapidly.Above-mentioned calculating is added in six orifice plates The virus quantity of gained adds the polybrene of final concentration of 8 μ g/m L,, will using ParafilmTM orifice plate after mixing well Orifice plate is placed in a centrifuge, and 800g centrifuges 60min；

4) orifice plate is taken out, orifice plate is placed in 37 DEG C, 5%CO₂Incubator in, continue culture for 24 hours；

5) 250g centrifuges 10min, removes containing virulent culture medium supernatant, and cell precipitation is resuspended with fresh culture, will Cell is transferred in six new orifice plates, continue culture 3-6 days it is spare；

(6) Western blot verify whether CAR structures are expressed

By detecting the CD3 ζ chains in CAR structures, to detect the expression of CAR in cell.

1. the extraction and preparation of total protein of cell sample

1) cell, including untreated T cell and infected T cell are collected；

2) add cell pyrolysis liquid RIPA into cell according to cell concentration, carefully blow and beat mixing with pipette tips, place on ice 10min；

3) whether observation liquid is relatively limpid, if color is partially yellow, can mend appropriate RIPA, is vortexed after concussion mixing, continues at 10min is placed on ice；

4) step (3) is repeated；

5) after cell fully cracks, 12000rmp, centrifuges 10min by 4 DEG C；

6) supernatant is collected, albumen concentration is surveyed using BCA methods, BCA kits are holy purchased from assist；

7) it is that 10 μ L determine protein sample sample-adding amount, while 2 μ L5 × loading Buffer are added with final applied sample amount, With Free H₂10 μ L of O polishings make final total protein concentration boil 5min for 100 DEG C after 20ng sealing membrana oralis；

8) residual protein sample preserves after 5 × loading Buffer can be added.- 20 DEG C can save one week, and -80 DEG C can protect It deposits one month；

2.Western blot detections

1) glue is fixed with clip, electrophoresis liquid is added thereto, it is ensured that no leakage；

2) electrophoresis liquid is added into electrophoresis tank；

3) vortex oscillator mixing and of short duration centrifugation are used before protein sample loading；

4) loading：Per 10 μ L of hole；

5) electrophoresis：80V, 30min；Turn 120V, until terminating；

6) transferring film：Appropriately sized pvdf membrane is cut, impregnates a few minutes activation in methanol liquid in advance.According to filter paper- The sequence of film-glue-filter paper is put well, is carried out electricity and is turned, and condition is wet turn, constant current 400mA, 1h；

7) it closes：5% skim milk, room temperature close 1h (or 4 DEG C overnight)；

8) PBST washes film：5min/3 times；

9)Anti-CD3zeta antiboby(1:1000) it is incubated, 4 DEG C overnight；

10) PBST washes film：5min/3 times；

11)HRP-labeled Goat Anti-Rabbit IgG(1:2000) it is incubated, room temperature 1h；

12) PBST washes film：5min/3 times；

13) it develops the color and takes pictures；

14) PBST washes film：5min/3 times；Appropriate membrane regeneration liquor is added, washes film 1h；

15) closing is re-started, and is incubated Beta Actin Antibody (1:4000)、HRP-labeled Goat Anti-MouseIgG(1:2000) it, develops the color, observation internal reference β-actin；

3. cytotoxicity analysis detects

Techniqueflow chart is shown in that Fig. 2, concrete operations flow are as follows：

1) detection plate is set, while setting 4 controls：Target cell maximum release group, volume correction control group, ground control group With Spontaneous release group, experimental group is arranged by 20: 1,10: 1,5: 1,1: 1 (effector cell: target cell).Target cell selects SK-HEP- (Western blot detect its GPC3, CD133 expression to 1 cell line, targeting antigen CD133 tables in SK-HEP-1 cell lines It is as shown in Figure 3 up to situation)；

A. effector cell hole is set up：+ 50 μ L culture mediums of 50 μ L effector cells；

B. experimental group：Target cell is constant, changes effector cell：+ 50 μ L target cells of 50 μ L effector cells；

C. the spontaneous release group of target cell is set up：+ 50 μ L culture mediums of 50 μ L target cells；

D. target cell maximum release group is set up：+ 10 μ L lysates (10 ×) of+50 μ L culture mediums of 50 μ L target cells；

E. volume correction control group is set up：+ 10 μ L lysates (10 ×) of 100 μ L culture mediums；

F. ground control group is set up：100 μ L culture mediums；Target cell inoculation number purpose optimizes

2) cell cracking and harvest supernatant：Every 100 μ L culture mediums add 10 37 DEG C of μ L cracked solutions (10 ×), 5%CO₂It is incubated 45min, 250g centrifuge 4min；

3) 50 μ L supernatants of transfer are protected from light the detection buffer solution for taking 12mL to thaw, by remaining rapid jelly to another orifice plate It deposits and (can be thawed, but can not be placed for a long time with 37 DEG C of water-baths).12mL detection buffer solutions are added to one bottle of Substrate cocktail In (can be used for two 96 orifice plates), it is inverted mixing；

4) diluted Substrate cocktail is added in 50 holes μ L/, and room temperature is protected from light incubation 30min, and (substrate is mixed after not used dilution Close thing liquid and be put in -20 DEG C, can save 6-8 weeks), add 50 μ L stop baths；

5) bubble removal that will contain in hole, the interior detection absorption values (490 or 492nm) of 1h, at least detects absorption value twice；

6) result counts

It is flat that all experimental group, the spontaneous release group of effector cell, the absorption value of the spontaneous release group of target cell should subtract background Equal absorption value；The absorption value of target cell maximum release group should subtract the mean absorbance of volume correction control group.Value after correction Statistics for killing rate：

Cell killing rate (%)=[(the spontaneous spontaneous release of release-target cell of experimental group release-effector cell)/(target cell The maximum spontaneous release of release-target cell)] × 100%.

As a result there are two types of manifestation modes：The intuitive of naked eyes is observed and reflects cell killing rate by measuring LDH；

Visually observe that (each group target is thin when bed board as it can be seen that experimental group target cell numbers are reduced compared with the spontaneous release group of target cell Born of the same parents' number is identical, uniform bed board), and with experimental group effector cell：The increase of target cell ratio, target cell numbers reduce brighter It is aobvious, it is lethal to illustrate that effector cell has target cell.

In addition, by the measurement of LDH, it can reflect cell killing rate, by drawing effector cell：Target cell-cell kills Hinder rate line chart, it can be seen that the two is positively related relationship.

The above result shows that the CAR-T cells obtained through slow-virus infection, it can be by expressing and identifying for tumour cell The antibody of the specific antigen on surface, the specific killing tumour cell.

The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be the present invention's Within protection domain.

Sequence table

<110>The Shanghai bio tech ltd Yi Hao

<120>The double target spot CAR-T therapy vectors and its construction method of liver cancer and application

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

tgccgcggcg acaagggccc cgactgc 27

<210> 2

<211> 789

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

ggcccaggcg gccgagctcg acattgttct ctcccagtct ccagcaatca tgtctgcatc 60

tccaggggag aaggtcacca tatcctgcag tgccagctca agtgtaagtt atatgtactg 120

gtaccagcag aaccaggatc ctcccccaaa ccctggattt atcgcacatc caacctggct 180

tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 240

agcagcatgg aggctgaaga tgctgccact tattactgcc agcagtatca tagttaccca 300

cccacgttcg gtgctgggac caagctggag ctgaaatcct ctggtggcgg tggctcgggc 360

ggtggtgggg gtggttcctc tagatcttcc ctcgaggtga agctggtgga gtctggacct 420

gagctgaaga agcctggaga gacagtcaag atctcctgca aggcttctgg ttataccttc 480

acagactatt caatgcactg ggtgaatcag gctccaggaa agggtttaaa gtggatgggc 540

tggataaaca ctgagactgg tgagccatca tatgcagatg acttcaaggg acggtttgcc 600

ttctctttgg aaacctctgc cagcactgcc tatttgcaga tcaacaacct caaaaatgag 660

gacacggcta catatttctg tgctaccgat tacggggact actttgacta ctggggccaa 720

ggcaccactc tcacagtctc ctcagccaaa acgacacccc catctgtcac tagtggccag 780

gccggccag 789

<210> 3

<211> 75

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcgggtgg aggcggttca 60

ggcggaggtg gctct 75

<210> 4

<211> 345

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180

agtcagaagt tcaagggcag agtcacgctg accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac aagattctac 300

tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 345

<210> 5

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45

<210> 6

<211> 339

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca gatctagtca gagccttgta cacagtaatg ccaacaccta tttacattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc caaccgattt 180

tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgct ctcaaaatac acatgttcct 300

cctacgtttg gccaggggac caagctggag atcaaacgt 339

<210> 7

<211> 204

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttac 204

<210> 8

<211> 123

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 9

<211> 126

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 10

<211> 336

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

agagtgaagt tcagcaggag cgcagagccc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 11

<211> 75

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

ggaggcggag gcgggagagc agaagggaga ggaagcttgc taacctgtgg agacgttgag 60

gaaaatccag ggcca 75

<210> 12

<211> 297

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60

agtggcacca atgatgctga agactgctgc ctgtctgtga cccagaaacc catccctggg 120

tacatcgtga ggaacttcca ctaccttctc atcaaggatg gctgcagggt gcctgctgta 180

gtgttcacca cactgagggg ccgccagctc tgtgcacccc cagaccagcc ctgggtagaa 240

cgcatcatcc agagactgca gaggacctca gccaagatga agcgccgcag cagttaa 297

Claims

1. a kind of Chimeric antigen receptor, which is characterized in that including CD133 single-chain antibodies, GPC3 single-chain antibodies and chemotactic factor (CF) Tri- specificity structures of CCL19,

Wherein, CD133 single-chain antibodies nucleotide sequence is as shown in SEQ ID NO.2,

GPC3 single-chain antibodies, by GPC3 single-chain antibody heavy chains V_H, Inter-Linker, GPC3 single-chain antibody light chain V_LThree parts structure At, wherein GPC3 single-chain antibody heavy chains V_HNucleotide sequence as shown in SEQ ID NO.4, the nucleotide of Inter-Linker Sequence is as shown in SEQ ID NO.5, GPC3 single-chain antibody light chains V_LNucleotide sequence as shown in SEQ ID NO.6；

Chemokines CC CL19 nucleotide sequences are as shown in SEQ ID NO.12.

2. a kind of Chimeric antigen receptor according to claim 1, which is characterized in that Chimeric antigen receptor structure composition is Igkappa-iRGD-CD133scFv-(G₄S)₅-GPC3V_H-(G₄S)₃-GPC3V_LscFv-CD8α-CD28-CD137-CD3ζ- T2A-CCL19。

3. a kind of Chimeric antigen receptor according to claim 2, which is characterized in that

IRGD nucleotide sequences as shown in SEQ ID NO.1,

CD133scFv indicates CD133 single-chain antibodies,

(G₄S)₅The hinge Inner-Linker between single-chain antibody, nucleotide sequence as shown in SEQ ID NO.3,

GPC3V_HIndicate GPC3 single-chain antibody heavy chains V_H, (G₄S)₃Indicate Inter-Linker, GPC3V_LScFv indicates that GPC3 is single-stranded Antibody light chain V_L,

CD8 α be transmembrane region, connect extracellular antigen binding domain and intracellular signal domain, nucleotide sequence as shown in SEQ ID NO.7,

CD28-CD137 is costimulation structural domain, and CD28 nucleotide sequences are as shown in SEQ ID NO.8, CD137 nucleotide sequences As shown in SEQ ID NO.9,

CD3 ζ be signal transduction domain, nucleotide sequence as shown in SEQ ID NO.10,

T2A is linker, and nucleotide sequence is as shown in SEQ ID NO.11.

4. a kind of double target spot CAR-T therapy vectors of liver cancer, which is characterized in that described embedding comprising any one of claim 1-3 Close antigen receptor and carrier.

5. double target spot CAR-T therapy vectors of liver cancer according to claim 4, which is characterized in that the carrier includes PUC19 Plasmid, Lentiviral.

6. double target spot CAR-T therapy vectors of liver cancer according to claim 5, which is characterized in that the slow virus expression carries Body is pCDH-EF1 α-MCS- (PGK-Puro).

7. a kind of construction method of double target spot CAR-T therapy vectors of liver cancer as described in claim 5 or 6, which is characterized in that

By Chimeric antigen receptor structure according to gene order, is synthesized using standard biologic synthetic method, be present in after synthesis On PUC19 plasmid vectors；PUC19 plasmid vectors and Lentiviral are all made of ECoR I, I double digestions of Not, by digestion Product is detached by agarose gel electrophoresis, is recycled purpose band, is obtained the concentration of carrier and target fragment, by the two according to rubbing You are than being 1:5 are attached conversion, and plasmid extraction is final to obtain the recombinant plasmid containing Chimeric antigen receptor structure, i.e., described Double target spot CAR-T therapy vectors of liver cancer.

8. a kind of application of double target spot CAR-T therapy vectors of liver cancer as described in claim 5 or 6, which is characterized in that be used for structure CAR-T cells are built,

The therapy vector is subjected to slow virus packaging using three plasmid packaging systems, group is as PSPAX2, pMD2G, pair of liver cancer Target spot CAR-T therapy vectors collect virus liquid, by this virus liquid using 293T cells as the incasing cells of slow virus, culture After concentration, T cell is infected, you can obtain CAR-T cells.

9. the application of double target spot CAR-T therapy vectors of liver cancer according to claim 8, which is characterized in that PSPAX2, PMD2G, liver cancer double target spot CAR-T therapy vectors have fixed content ratio in system.

10. a kind of CAR-T cells, which is characterized in that be prepared using the application process of claim 8.